PhD defence: Breaching Barriers in Gene Editing: Getting CRISPR/Cas9 Ribonucleoproteins into the Cell with Cell Penetrating Peptides

The CRISPR/Cas system is a powerful tool for both scientific research and potential therapeutic strategies for inherited diseases. This PhD thesis investigated the feasibility of cell-penetrating petide-mediated direct delivery of CRISPR/Cas components to achieve various CRISPR/Cas-based gene editing applications. In order to reduce off-target effects that result from long-term high expression and activity of CRISPR/Cas9, rather than employing mRNA or plasmid platforms of Cas9 as delivery format, this work focused on the direct delivery of the Cas9 ribonucleoprotein complex.

After screening a variety of cell-penetrating peptide sequences, we uncovered a highly efficient delivery of Cas9 using the amphipathic LAH5 peptide. This peptide sequence showed a robust capacity to form complexes with Cas9 ribonucleoprotein, even with an additional single-stranded DNA homology directed repair (HDR) template which can be used to generate specific genetics repairs or mutations. The nanocomplexes formed with the Cas9 ribonucleoprotein and the LAH5 peptides were effectively taken up by cells, resulting in effective gene editing and correction in a variety of human cell types. Whereas cell-penetrating peptides have been shown to facilitate efficient transfected of various cargos in an in vitro setting, these systems have various limitations that may hamper therapeutic applications, such as decreased stability in the presence of serum. To address these limitations, we studied the effects of N-terminal acylation with various fatty acids on the capacity of LAH5 peptides to form nanocomplexes with Cas9. Furthermore, we studied the overall stability of such nanocomplexes, as well as their protective capabilities against protein degrading enzymes, their capability of disrupting cell membranes, and their efficiency in Cas9 delivery. Fatty acid modifications, especially Oleic acid provided increased durability against proteases and increased stability in serum rich conditions and, more importantly, Oleic acid-LAH5/Cas9 nanocomplexes showed increased capability of facilitating effective gene editing and correction in various cell types.

To further study the potential therapeutic applications of these peptides, their potential to correct pathogenic mutations was studied. Using the described Oleic acid-LAH5 peptides, a CRISPR/Cas9 mediated editing strategy was used to eliminate multiple splicing defects that have been shown to cause Stargardt disease (STGD1). This rare genetic disease that results in vision loss is caused by deep intronic (DI) variants within the same intron of the ABCA4 gene. Ultimately, our strategy led to the restoration of normal ABCA4 gene splicing and expression levels in derived pluripotent stem cell (iPSC) cell models. In a further study, nanocomplexes were tested in vivo and successfully delivered Cas9 to muscle tissue, achieving effective gene editing in skeletal muscle.

Altogether, our findings demonstrate the potential of LAH5 peptide-mediated efficient gene editing in a wide variety of cell types under in vitro conditions, as well as local in vivo gene editing. These findings could open the door to the potential development of a variety of potential gene therapy-based applications such as ex vivo gene editing, or future treatments of genetic pathologies in muscle tissues. Additionally, the findings in this thesis provide a versatile, and highly efficient delivery method for Cas9, which could be utilized as a robust tool for biomedical research in future studies. The discoveries in this thesis provide new insights that have the potential to benefit the design and application of novel delivery vectors for future therapeutic strategies to treat genetic diseases.